SOME ASPECTS OF NUCLEIC ACID

iA2>TD PROTEIN SYNTHESIS IN CELL NUCLEI

DISSERTATION

Presented in Partial Fulfillment of the Requirements for -trhe Degree Doctor of philosophy in the Graduate School of The Ohio State University

By TEiEODORE RONALD BREITMAN, B. S., M. S.

******

The Ohio State University 1958

Approved by

dviser Department of Agricultural Biochemistry Ack no w 1 e dgme n t s

The author wishes to express his appreciation to

Dr. George C. Webster for his counsel and guidance throughout the course of this investigation.

A special note of thanks goes to Dr. Ruth G.

Kleinfeld for her collaboration in the thioacetamide experiments. iii

Table of Contents

Page

Introduction ...... 1

Studies on Nuclear Metabolism in vivo ...... 1 a. Ribonucleic Acid...... 1 b. Deoxyribonucleic Acid...... 4 c. Protein...... 6

Studies on Nuclear Metabolism in vitro.

a. Ribonucleic Acid...... eoaaaoaoooaoa* b. Deoxyribonucleic Acid...... 13 c. Protein...... 15

Aims of this Investigation...... 21

E xp e r 1 me ntalo..ooo...... ««.««.«..«.oo«23

Methods...... 23

Results ...... 25 Effects of Monovalent Cations...... 25 Time Course of B 3* Incorporation into RNA...... 31 Inhibitor Studies...... 31 The Effect of Thioacetamj.de on Nuclear RNA Metabolism in vivo...... 41 Effect of Thioacetamide on Isolated Thymus Nuclei...... 46

Discussion..eo.o..o.oo«.....o.ooo.ooo...... 48

Summary...... «o.. o..oo.o q . o »o.o.oooo. o.... oO

Bibliography...... ooo....oooo.o.oo.ooo.oo.ooo2

A.U tOb lO gr aphy oo«o«ooo....ooooo>oe.«*.oo.oo.o.ooooo»...35 iv

List of Tables

P age

Table 1. Incorporation of the Carbon or Phosphorus of Various Labeled Compounds into Nuclear RN'A, DiSfA, or Protein...... 25

Table 2. Effect of Replacement of Sodium by Potassium Ions on the Incorporation of Various Precursors into Nuclear RNA, DNA, and Protein...... 27

Table 3. Effect of Partial Replacement of Sodium Ions by Various Monovalent Cations on Glycine Carbon Incorporation into RMA, DNA, and Protein...... 30 V

List of Figures

Page

Figure 1. Inhibition of the incorporation of glycine carbon into nuclear Rl\iA, DNA., and protein by progressive replacement of sodium by potassium ions...... 29

Figure 2. Time course of P incorporation into the iltJA of isolated thymus nuclei..... 32 1 A Figure 3. Effect of chloramphenicol on alanine-C incorporation into protein and ortho- phosphate-p32incorporation into RNA and DMA...... 34

Figure 4. Effect of purine and S-mereaptopurine on the incorporation of glycine carbon into nuclear RMA, DMA, and protein...... 33

Figure 5. Effect of amino acid analogs on the incorporation of glycine carbon into nuclear RIIA, DLSFA, and protein...... » ...... 37

Figure 5. Effects of ribonuclease and deoxyribo­ nuclease on the incorporation of glycine carbon into nuclear RjtfA* DMA, and protein. „......

Figure 7., Effects of varying concentrations of RiSl'Aase on the incorporation of glycine carbon into nuclear RNA and protein.... 40

Figure S. Specific activitv/DNA/RNA-tiiae curves for nuclear RNA...... 42

Figure 9. Specific activity/DNA/RNA-time curves for nuclear RNA...... ,, 44

Figure 10. Effect of TA on the DNA/RNA ratios...... 45

• i 4 Figure 11. Effect of TA on alanine-CJ" incorporation into the protein of isolated thymus nuclei...... 47 1

Introduction

The cell nucleus, because of its active role in cellu­ lar division and its presence in most animal and plant cells, has been actively studied by the cytologist for some time. The biochemist's interest in events taking place in the nucleus was seriously hampered by the lack of techniques for both isolating and analyzing the nucleus for its constituents. The advent of methods for breaking up tissue and isolating cellular components has made possible, as with other cellular entities, the more intimate mode of investigation that we are familiar with today.

It would be redundant to review here the vast number of papers dealing with the biochemistry of the nucleus.

Many excellent reviews are available which treat the vari­ ous areas of this growing field (1-7). It would, however, be within the scope of this introduction to review briefly those aspects of nuclear metabolism which bear closely to the contents of this dissertation.

Studies on Nuclear Metabolism in vivo

a. Ribonucleic Acid (RNA)

With the availability of isotopes, it became possible to study intermediary metabolism at a level of sensitivity never before thought possible. Such studies using a vari­ ety of isotopically labeled precursors (e.g., phosphate la- 32 14 beled P , C -formate and a diversity of amino acids, pu­ rines, pyrimidines, nucleotides, and nucleosides labeled 35 15 with radiocarbon, tritium, S , and/or N ) have made it clear that the RNA of the nucleus has a marked metabolic 32 activity. In 1948 Marshak (8), using P and Bergstrand 15 et al. (9), using glycine-N , reported that more of these isotopes were incorporated into the nuclear RNA than was taken up by the cytoplasmic RNA, an observation which has since been confirmed in many other laboratories using a variety of isotopes and materials (10-19). Such evidence led several workers to propose that the nuclear RNA is the precursor of cytoplasmic RNA (10,20). on the other hand, it has been proposed by Barnura et al. (12,13) that the nucleus and cytoplasm synthesize RNA independently, the nuclear rate being higher. Another objection to the pro­ posed precursor role of nuclear RNA is based on the ob­ served differences in the nucleotide composition of nuclear

RNA and cytoplasmic RNA when compared in bulk (21-23) .

This would appear to obviate the possibility that nuclear

RNA could simply diffuse into the cytoplasm. More recently, it has been reported that a nuclear RNA fraction, soluble in neutral phosphate buffer, has the same nucleotide compo­ sition and electrophoretic mobility as that of cytoplasmic 32 RNA and incorporates p into its mixed nucleotides at a substantially greater rate than does the corresponding cytoplasmic RNA (24,25). However, these results are not entirely in accord with an earlier paper of Logan and Davidson (26) who report differences in composition between the soluble nuclear RNA and the cytoplasmic RNA.

Other lines of research have drawn attention to the dependence of cytoplasmic RNA on the activity of the nuc­ leus as shown by comparisons between nucleated and enuc­ leated cells. The researches of Linet and Brachet (27) and of James (28), on Amoeba. agreed in the observation that the

RNA level declined in the enucleated portions but tended to be maintained in the nucleated fragments. The results ob­ tained by Goldstein and Plaut (29) clearly demonstrated the transfer of nuclear RNA or its components to the cytoplasm, and gave no indications for the reverse process. Further experiments by Plaut and Rustad (30) indicated that adenine- 14 8-C was incorporated into the RNA of the enucleated por­ tion of Amoeba at a slower rate than into the nucleated portion or into the whole organism. In contrast to these results with Amoeba fragments, which have been interpreted as strongly suggesting a nuclear control on cytoplasmic RNA metabolism, are the results of Brachet and coworkers (31,

32) who have demonstrated in Acetabularia that enucleate fragments can grow, synthesize protein and RNA, and differ­ entiate without a nucleus. These authors have also found that both enucleate and nucleate portions of Acetabularia 14 could incorporate orotic acid-C into their RNA, the nucleated half incorporating 1.4 times as much label as the enucleated half. A more critical experiment on Acetabularia has recently been reported by Stich and Plaut

(33). Ribonuclease treated nucleate and enucleate frag­ ments of Acetabularia were investigated for their ability

to grow, synthesize protein, and differentiate. It was

found that while nucleate portions recovered their capacity

in these respects, enucleate fragments did not. The au­

thors have interpreted these results to indicate that some

nuclear product was required for recovery and that the product is most probably nuclear RNA.

While the results outlined above leave little doubt

for the existence of a nuclear mechanism for RNA synthesis,

the metabolic role of this constituent in the nucleus and

its possible role in other parts of the cell has still to be clearly elucidated. The possibility that RNA or ribo-

nucleoprotein plays a role as a mediator of genetic infor­ mation exists (34). Experiments with viruses demonstrate

that RNA may in some situations transmit genetic informa­

tion (35,36) .

b. Deoxyribonucleic Acid (DNA)

It has become a generally accepted observation that

DNA is a metabolically stable cellular component, the syn­

thesis of which occurs principally in connection with cel­

lular division. The biochemical researches which have led

to this concept were initiated by Hevesy in 1940 (37) and

further confirmed and extended in the years immediately following by Brues et al. (38) and Marshak (8) . These in- 32 vestigators showed that the incorporation of P -labeled

phosphate into the DNA of various tissues closely paral­

leled the rate of cell division. In resting tissues the 32 rate of P incorporation into DNA-phosphorus is very small 32 but the incorporation of P increases in proportion to the

mitotic index of growing tissues (39,40). Correlative with

these findings has been the observation that once formed

the DNA molecule appears to be stable (41-44). using

regenerating liver and labeled adenine Bendich (41) has

calculated a "half-life" of about 50 days for DNA as com­ pared to about 8 days for RNA.

In recent years the concept has been advanced that

metabolically and functionally different DNA's are present

in the same set of chromosomes (11, 45-50) . Of significance

are the findings of Bendich and associates (51,52) with

respect to the fractionation patterns of DNA in different

tissues of the same organism, on the basis of chromato­

graphic profiles, one can distinguish the following kinds

of DNA: calf thymus, human leukemic leukocyte, various bac-

teria, T2r, T6r,and T6r phage, spleen, intestine, and

brain of the rat. In the case of the T6r phage strains, a

single genetic change is involved, and a difference in

chromatographic profile is seen. The various DNA molecules

in a given cell may differ among themselves not only in

composition (48) and sequence but also in shape, size, and metabolic activity (53).

c. Protein

That the nucleus might play a vital role in the process of protein synthesis was proposed in 1940 by

Caspersson (54-56) as part of his general theory of protein synthesis in living cells, caspersson proposed his theory on the basis of results obtained by direct chemical analy­ sis and his method of ultraviolet microspectophotometry.

Another very important aspect of this theory was the intro­ duction of the concept that protein synthesis was dependent on the presence of nucleic acids. In tribute to Caspersson it should be stated that to a surprising extent his theory has stood the test of time. 15 The results of Daly et a].. (57) , using glycine-N , and 14 of Smellie et. ad. (58,59), working with formate-C , me- 35 15 thionine-S , and glycine-N , indicate that the incorpo­ ration of these substances into nuclear protein and cyto­ plasmic protein is of the same magnitude. These results would appear to indicate that in liver tissue the nucleus might be the main site of protein synthesis. However, in these experiments the cytoplasmic proteins were not frac­ tionated. In experiments where labeled amino acids were injected intravenously into animals or administered to intact bean hypocotyls the highest incorporation was in the microsomes (58,60-66). Unfortunately, no experiments have been reported in which nuclei have been fractionated,

as has been the cytoplasm, into various protein containing

components to be compared with the cytoplasmic particles

for amino acid uptake. There is reason to suspect on the basis of the results of Daly et al. (57) and experiments in vitro to be discussed in a later section, that the RNA-

and DNA-associated proteins of the nucleus may have a pro­

tein synthesizing capacity comparable to the microsome

fraction of the cytoplasm. The autoradiographic experi­ ments of Ficq (67) and Ficq and Errera (68) have shown a higher uptake of glycine by nuclear than by cytoplasmic pro­

teins, suggesting the possibility that during the isolation

of nuclei from whole tissue, some highly labeled protein may have been lost in the experiments of Daly et al. (57)

and of Smellie est al. (58,59) mentioned above.

The finding that non-nucleated cells such as reticulo­

cytes incorporate labeled amino acids into the heme moiety

in contrast to mature erythrocytes which do not have this

capacity (69-71) would appear to indicate that the nucleus

is not involved with cellular protein synthesis. Further­

more, Holloway and Ripley (72) have shown that during the

development of reticulocytosis there is a considerable in­

crease in the RNA content along with a parallel increase in

the amount of labeled leucine incorporated into the pro­

teins. These results indicate a close link between RNA and protein synthesis and give no suggestion of a direct role played by the nucleus.

That protein synthesis can proceed without the nucleus has been definitely established by a number of workers us­ ing the green alga, Acetabularia mediterranea. As early as

1934 Hammerling (73) had observed that an enucleated half could regenerate to a fairly large extent. Vanderhaeghe

(74) quantified these results. He found that the regener­ ation was accompanied by increases in dry weight and pro­ tein and that the rate of regeneration was the same in both enucleated and nucleated fragments for two weeks, at which time it slowed down and finally stopped after sever­ al months in the enucleated portion. Experiments using radioactive precursors have confirmed Vanderhaeghe's obser­ vations and in addition have uncovered two indigenously cytoplasmic protein synthesizing systems, the chloroplasts which are capable of synthesizing protein from CC>2 only in the presence of light (77) and the microsomes which incor­ porate glycine into protein irrespective of light (75-77).

The complete lack of direct nuclear control on protein syn­ thesis in this system is quite apparent. However, the recent investigations of Stich and Plaut (33), previously discussed, would indicate that nuclear control could be exerted indirectly by means of the assimilation of nuclear

RNA into the cytoplasm. On the other hand, it is difficult to understand how an enucleated fragment could continue to carry on its functions in a normal manner for two weeks if nuclear RNA were required. A more reasonable explanation might be that some functions, e.g., synthesis of various enzymes, would be under the direct control of nuclear RNA while others would be under the control of the RNA synthe­ sized in the cytoplasm. That this may be the case is indi­ cated by experiments reported by Brachet and his associates

(78,79) who have studied the adaptive synthesis of catalase in nucleated and enucleated fragments of Acetabularia.

They have found that the induction of increased catalase activity on addition of hydrogen peroxide can be demon­ strated in nucleated portions for 1 week before it slows down, while enucleated fragments retain this capacity for three months.

Studies on Nuclear Metabolism in vitro

a. Ribonucleic Acid (RNA)

The synthesis of nuclear RNA ini vitro has been studied in Ehrlich ascites cells (81-83), rabbit bone marrow (81) , regenerating rat liver (82-84), various embryonic tissues

(82,83), isolated nuclei (85-88), and in liver homogenates

(89). Because of its relevancy to this dissertation, the work which has been conducted on isolated nuclei will be stressed.

Studies on nuclear nucleic acid metabolism in vitro were initiated by Siebert et al. (85) in 1953. These 10 workers employed isolated pig kidney nuclei and studied the 32 incorporation of P into the phosphorus-containing com- 32 pounds. Though incorporation of P into the nuclear nucleic acid was observed, several of the experimental findings would tend to cast some doubt as to the validity 32 of these results. Maximum P uptake was observed in only

10 minutes and the optimum temperature for P 32 incorpora­ tion was 20°C. It would be difficult to reconcile these results with those of other workers (87,88) who have found that the incorporation of several precursors into RNA pro­ ceeds for several hours at 37°C.

Two distinct RNA components have been described in nuclei (24,26,87,88) which differ mainly in their meta­ bolic activity, one fraction is obtained by extracting isolated nuclei with pH 7.1 phosphate buffer. This com­ ponent is called the "pH 7.1 soluble RNA" or RNA I. The residue remaining after 1M Naci extraction is termed "1M fraction,""nucleolar RNA" or RNA II. In experiments in vitro it has been shown that there is a marked difference in the uptake of adenine (88), orotic acid, and adenosine

(87) by the two nuclear RNA's. These RNA precursors are rapidly incorporated into RNA II while incorporation into

RNA I is from 80-90% less. Thus the results in vitro are in good agreement with the experiments _in vivo previously discussed (24,26). Both Logan and Davidson (26) and osawa et ail. (24) have noted differences in the molar proportions 11 of the bases in RNA I and RNA II. Allfrey and Mirsky (87) however, report essentially the same composition for these two RNA fractions.

Surprisingly little work has been done to elucidate the intermediate synthesis of nuclear RNA. The only re­ port worth mentioning in this connection is that the enzyme polynucleotide phosphorylase has been reported to be pre­ sent in guinea pig liver nuclei (90). This enzyme, iso­ lated from Azobacter vinelandii, has been extensively stud­ ied by Ochoa and coworkers (91). It catalyzes the synthe­ sis of highly polymerized ribonucleotides from 5'-nucleo­ side diphosphates. Except for the report of Hilmoe and

Heppei (90) this enzyme has been found only in microorgan­ isms and plant material (91). The finding of cannellakis

(92) that a dialyzed extract of an acetone powder prepared from the soluble fraction of rat liver is capable of incorporating adenylic acid from ATP into a RNA-like mate­ rial and his finding that ADP is not acted upon in this system is indicative that another enzyme system, other than polynucleotide phosphorylase, is operative in mammalian tissue. Cannellakis (92) further showed that the adenylic acid incorporated was linked to a monoesterified terminal cytidylic acid, therefore becoming a terminal monoesteri- fied nucleotide itself. These interesting findings of

Cannellakis have recently been confirmed and extended by

Hecht e_t al. (93) who have found that the nucleoside tri­ 12 phosphates of cytosine, adenine, and uracil are incorporat­

ed into the soluble RNA of Ehrlich ascites carcinoma cells

in vitro. In agreement with Cannellakis, Hecht ^t ad^. have

found that cytosine- and adenine-5'-nucleotides are present

in the RNA in sequence, the AMP being predominantly termin­

al. It has been pointed out by Hecht _et al. (93) that while this enzyme system differs from that reported by

Ochoa et ad.. (91) , in that it does not react with nucleo­

side diphosphates, an even more important distinction is

that no net synthesis of RNA is observed. The possibility

exists however, that under conditions different from those used by Hecht et al. a net synthesis of RNA may be exhib­

ited .

Logan and Smellie (89) have investigated the relation­

ships between the nucleic acids of rabbit liver nuclei and cytoplasm. They have found that when cytoplasm from ani- 32 mals previously injected with P is incubated with unla­ beled nuclei both the nuclear RNA and to a lesser extent

the DNA become labeled. In the converse experiment, where

labeled nuclei are incubated with "cold" cytoplasm, cyto­ plasmic RNA does not become labeled although there is a

considerable decrease in the activities of both the nuclear

RNA and DNA. Fractionation of the cytoplasm indicates that

the active factor and/or precursors are in the non-dialyz- able portion of the soluble phase. 13

b. Deoxyribonucleic Acid (DNA) 32 Siebert et. _al. (85) noted the incorporation of P into the DNA of isolated pig kidney nuclei. A more crit­ ical experiment was conducted by Friedkin and Wood (94), 14 who studied the utilization of thymidine-C by isolated thymus nuclei. These workers found that thymidine was incorporated into DNA whereas thymine was not. It was also observed that treatment with saponin, or freezing and thawing, inactivated the nuclei completely. Addition of deoxyribonuclease (DNAase) or versene to the incubation medium markedly inhibited thymidine incorporation into DNA.

However, the absence of potassium or magnesium ions in the media had only a slight inhibitory effect. The addition of a mixture of purine and pyrimidine bases, ribonucleosides, and ribonucleotides had no effect on thymidine incorpora­ tion. in contrast, mixtures of deoxyribonucleosides and deoxyribonucleotides were inhibitory. By testing the indi­ vidual deoxyribonucleosides it was found that only deoxy- cytidine was inhibitory. It is very likely that the appar­ ent inhibitory action of deoxycytidine is due to its de­ amination to uracil deoxyriboside (95-98) and then methyl- ation to a thymidine derivative (99,100) which is incorpo­ rated into DNA, tending to dilute out the labeled thy­ midine in the incubation media, chromatography of thymi­ dine-labeled DNA by the methods of Bendich et_ aJL. (101) yielded fractions differing in specific activities from 14

502 to 1350. Of especial interest is the finding that the most active DNA fraction was that one which was most diffi­ cult to dissociate from protein. The finding of Allfrey et al. (86,102) that the most active protein synthesis in iso­ lated thymus nuclei is associated with the deoxyribonucleo- protein makes this observation all the more significant.

In recent years great strides have been made in the elucidation of the mechanism of DNA synthesis. An enzyme system capable of incorporating deoxyribonucleotides into

DNA has been demonstrated in extracts of E . coli (103-107) and in homogenates and extracts of animal tissue (108-111).

The enzyme, polydeoxynucleotide pyrophosphorylase has been purified over 2000 fold by the Kornberg group (107) and it has been found that highly polymerized DNA, 4 deoxyribo- nucleoside triphosphates (adenine, guanine, cytosine, and thymine) and Mg are required for DNA synthesis (107).

These results with the E. coli enzyme system have been essentially confirmed using the high speed supernatant fraction of homogenates from rat liver (108-110). In rat liver this enzyme system is apparently • localized in the

100,000 x supernatant fraction (109,110). Mantsavinos and canellakis (110) have reported that this fraction from rat liver will incorporate deoxyribonucleotides into DNA, apparently without the presence of preformed DNA in the medium. These workers have also found that addition of rat liver DNA to the incubation medium increases incorpora­ 15 tion and that addition of all four nucleotides or an extract of regenerating rat liver nuclei increases nucleotide incor­ poration still more. In contrast, Bollum and Potter (109), in agreement with Kornberg, have demonstrated an absolute requirement for the presence of acceptor DNA in the incu­ bation media in order for new DNA synthesis to proceed. The source of DNA is apparently not too critical; calf thymus

DNA, salmon sperm DNA, or rat liver nuclei from normal or regenerating livers have been employed. It is of interest to note that no essential difference was noted in the incor- 3 poration of thymidine-H into the DNA of nuclei from normal or regenerating rat livers in the presence of the 100,000 x £ supernatant fraction. As mentioned previously, it has been reported that an extract from regenerating liver nu­ clei will increase the incorporation of guanosine triphos­ phate into DNA by a factor of 3 fold in a completely solu­ ble system (110).

The localization of the DNA synthesizing activity in the cytoplasm is a very interesting and surprising finding.

It would be expected that the enzymes involved with DNA synthesis would, as is DNA, be localized in the nucleus.

One possible explanation would be that this enzyme system is synthesized in the cytoplasm and can, under the control of a regulatory mechanism, diffuse into the nucleus. In cells in which rapid growth is occurring one might expect a greater diffusion of the enzyme. The fact that an ex- 16

tract front regenerating liver nuclei increases the rate of

incorporation of a precursor into DNA in a system in vitro

(110) might be rationalized by this explanation.

Recently, Sekiguchi and Sibatani (111) have made a

study of the effects of deoxyribonuclease treatment on the 32 incorporation of P into the nucleic acids of isolated

rabbit appendix nuclei. Their data indicate that the de­

creases in amount of DNA and the specific activities of both the RNA and DNA are essentially parallel. Removal of

70-80% of the DNA completely abolished the incorporation 32 of P into RNA and DNA. It was also found that when 64%

of the DNA was removed, the addition of yeast RNA, or salm- 32 on sperm DNA restored the incorporation of P into RNA to

control levels. However, DNA synthesis was restored to

60-70% of the control level. In contrast, when over 73%

of the DNA was removed, the addition of these compounds

exhibited very slight restoring power.

c. Protein

Studies on the incorporation of labeled amino acids

into isolated nuclei were first reported by Lang et al.

(112). The first extensive study of protein synthesis in

isolated nuclei was carried out by Ailfrey (113). In this

initial study, Ailfrey found that the incorporation of 14 alanine-C into the protein of isolated calf thymus nuclei

was appreciable when compared to the low uptakes reported by Lang ej; al. (112) using isolated pig kidney nuclei and

by Siekevitz (114) using liver homogenates. He further ob­

served that in the absence of an energy source (a-ketoglu-

tarate) the uptake of alanine was reduced by 40-75%. Of

prime concern was the observation that ribonuclease treat­

ment had no effect on protein synthesis, while deoxyribo­

nuclease treatment markedly inhibited amino acid incorpo­

ration. Addition of thymus DNA reversed this inhibition by

deoxyribonuclease. This finding that DNA, not RNA, is

essential for amino acid incorporation into nuclear protein

has been in sharp contrast with results obtained using

bacterial (115-118) and isolated cytoplasmic particulate

systems (119-122). In subsequent reports, Ailfrey et al.

(86,123,124) have further clarified and extended these

findings. The requirement for an energy source (a-keto-

glutarate) has been found to be in reality a requirement

for the sodium ions which were associated with this organic

salt. A final sodium ion concentration of 0.068 M has been

found to give maximum incorporation of amino acids into

nuclear protein. No requirement for potassium ions could be shown, probably because potassium ions are present in

the nucleus (125).

The effect of adding supplementary DNA to nuclei which

had had 70% or more of their DNA removed by deoxyribonucle­

ase indicated that calf thymus DNA does, in fact, restore

much, and at times, nearly all of the protein synthesizing 18 activity of the nucleus. The following DNA's or products of DNA have been found to have restoring capabilities: alkali denatured thymus DNA, apurinic acid, thymus DNA, core remaining after DNAase digestion of thymus DNA, dial- yzable products of DNAase digestion of thymus DNA, calf kidney DNA, chick erythrocyte DNA, Paracentratus sperm DNA, trout sperm DNA, and wheat germ DNA. Both the wheat germ

DNA and apurinic acid were about 50% as effective as the other compounds listed, of further interest is the finding that calf liver RNA, yeast RNA, and polyadenylic acid are as effective as calf thymus DNA in restoring amino acid in­ corporation into nuclear protein (126). However, alkaline digests of RNA, mixtures of nucleoside 2'- and 3 '-phos­ phates or ribonucleoside-51-phosphates, AMP, ADP, and a number of dinucleosides are inactive (86,126). The fact that RNA is active in this system is as yet unexplained.

Nor is it clear what function DNA plays in nuclear protein synthesis.

One function of RNA and DNA in isolated nuclei appears to concern the synthesis of adenosine triphosphate (ATP).

Ailfrey and Mirsky (126) have shown that nuclei deprived of

55-70% of their DNA, by the action of DNAase, lose almost all of their ability to synthesize ATP. Almost normal lev­ els of ATP could be restored by adding thymus DNA or yeast

RNA to a suspension of the DNAase treated nuclei. It is interesting to note at this point that two recent reviews 19 misinterpret the data and/or the interpretation of these results. Chantrenne states on p. 39 of his review {121),

...ATP production (after DNAase treatment is restored by the same substances which restore amino acid incorporation, namely DNA, split prod­ ucts of DNA,RNA, and polyadenylic acid. The observed effects of DNA are thus explained by its being involved-in some unknown way-in ATP form­ ation in isolated nuclei, and there is no evi­ dence left that DNA is directly involved in pro­ tein synthesis, even in the nucleus itself ...

Heppel and Rabinowitz on p. 624 of their review (128) state,

An influence of poly A (polyadenylic acid) and other polynucleotides on the ATP level of iso­ lated thymus nuclei has been reported.

Ailfrey and Mirsky's paper indicates that only DNA and

RNA were tested. The effect of removing RNA was not inves­ tigated in terms of ATP synthesis, but the observation that the addition of RNA will induce the synthesis of ATP in DNA-depleted nuclei indicates that this polynucleotide, as well as DNA, plays some role in ATP synthesis. More recently, it has been reported by Ailfrey and Mirsky (129) that the addition of a number of polyanions (e.g., heparin, chondroitin sulfate, and polyethylene sulfonates) to nuclei deprived of 70% of their DNA will restore as much of the nuclear ability to synthesize ATP and to incorporate pre­ cursors into protein and RNA as added RNA and DNA. Poly­ cations had no effect, While these results may be inter­ preted as indicating that the polyanionic nature of DNA is responsible for its involvement in ATP, nucleic acid, and 20 protein synthesis, the evidence linking DNA with a genetic role and therefore a function in the synthesis of specific macromolecules cannot be overlooked. One cannot refrain from questioning whether or not functional RNA and protein are being synthesized when polyanions are added to DNA de­ pleted nuclei. Experimentally, this poses a difficult question to answer since so little protein and RNA are synthesized by isolated nuclei, one possible approach to this problem might be to study a specific protein known to be synthesized in the nucleus, such as diphosphopyridine pyrophosphorylase, and to see how its synthesis is affected by DNAase treatment and the addition of various polyanions.

A number of inhibitors have been tested by Ailfrey et al. (36) for their abilities to inhibit protein synthesis.

Chloramphenicol, and the amino acid antagonists, p-fluoro- phenylalanine and ethionine, had little effect on the in­ corporation of amino acids. In contrast, it was found that cortisone and 5,6-dichloro-j3-D-ribofuranosylbenzimidazole

(DRB) led to greatly reduced alanine uptakes. It was fur­ ther shown that DRB would inhibit protein synthesis only when present in the incubation medium at "0" time. The synthesis of RNA was inhibited at anytime by DRB. Un­ fortunately the effect of DRB on DNA synthesis was not in­ vestigated.

Fractionation of the nuclear proteins after incubation 14 with alanine-C have indicated that the protein most closely associated with DNA is more active than all other proteins of the nucleus, with the occasional exception of the small fraction extractable in pH 7.1 phosphate buffer

(RNA I) (86). As previously discussed, Friedkin and Wood

(94) have reported that the most active DNA synthesis oc­ curs in a DNA fraction closely associated with protein. A close relationship between DNA and protein synthesis may be indicated by these observations, in contrast, the same picture does not seem applicable to RNA and protein syn­ theses since it has been established from the results of experiments both jLn vitro (87,88) and iri vivo (24,26) that

RNA II is markedly more active than RNA I.

Aims of this investigation

At the commencement of this study little concrete in­ formation was available concerning the mode of biosynthesis of protein and nucleic acid in cell nuclei, and of the pos­ sible relationship of these processes. The initial reports of Allfrey et al. (102,113), indicating that isolated nu­ clei could be employed as experimental material for the study of protein synthesis, demonstrated that the relation­ ship of protein and nucleic acid synthesis could be ap­ proached with isolated nuclei.

In regard to these relationships it was of interest to determine what effects monovalent cations and various known nucleic acid and protein inhibitors would have on 22 these synthetic processes. It 'was also of some concern to investigate the capability of isolated nuclei to incorpor- ate a number of known nucleic acid precursors.

Another phase of this study is concerned with the ac­ tion of the drug, thioacetamide (TA). Recent investigations have indicated that considerable increases in the nuclear

RNA and protein content could be induced in the liver and kidney cells of rats on administration of this drug (130,

131). Typical morphological and biochemical changes are observed in less than two days (131) . These changes in­ clude increased nuclear, nucleolar and cell volumes, in­ creased levels of nuclear RNA and protein, and a diffuse distribution of cytoplasmic ribonucleoprotein (130-132) .

The observed increase in nuclear RNA (primarily with­ in the nucleoli) during TA treatment appears to be a rapid and specific response. It was felt that three general possibilities could be offered in postulation of the nature of this increase: (a) an increase in nuclear RNA synthesis is induced, (b) a decrease or blockage of nuclear RNA breakdown or assimilation to the cytoplasm results or

(c) a combination of (a) and (b) . 23

Experimental

Methods

The procedures for the isolation and incubation of calf thymus nuclei were those of Allfrey et _al. (86) . One ml of nuclear suspension was added to a medium consisting of 0.5 ml 0.1 M sodium or potassium phosphate buffer in

0.25 M sucrose, 0.4 ml sodium chloride-0.1 M glucose containing 6.25 mg Naci/ml, and 0.1 ml radioactive tracer.

Inhibitors were dissolved in the sodium chloride-glucose solution at 5 times the desired final concentration. For the nuclease experiments, the crystalline enzymes were dissolved in water and 0.1 ml of this solution was added to the reaction mixture minus the radioactive tracer. For both the inhibitor and nuclease experiments, the nuclei were pretreated for 30 minutes at 38°C in a metabolic shak­ er at which time 0.1 ml of the tracer was added and incu­ bation then continued for two hours. The chloride salts of Rb, Cs, K, Li, and NH^ were substituted in varying amounts for the sodium chloride in the sodium chloride- glucose solution in those experiments designed to study the reported sodium requirements of isolated nuclei. In the case of K, potassium phosphate buffer was used instead of sodium phosphate buffer.

At the conclusion of the incubation period, the con­ tents of the incubation vessels were quantitatively trans­ 24

ferred to 15 ml conical centrifuge tubes with cold 2%

perchloric acid. The tubes were filled to 15 ml with 2%

perchloric acid and centrifuged in the cold. The precipi­

tate was then washed 4 times with 2% perchloric acid, 2

times with hot 95% ethanol, 2 times with an ethanol-ether

mixture (3:1), and finally once with ether.

The nucleic acids and protein were separated by a

modified Schmidt-Thannhauser (133) procedure using the

hydrolysis procedures of Kleinschmidt and Manthey (134).

The hydrolyzed RNA and DNA were either assayed direct­

ly or first purified by adsorption on and elution from

Dowex 1 (135) and then measured at 260 m|i in a Beckman

spectrophotometer using an absorbancy index of 32.1 and

26.1 for the RNA and DNA, respectively. Protein concentra­

tion was measured by the biuret reaction (136).

Aliquots of the hydrolyzed RNA and DNA and protein

were dispersed evenly on glass planchets and assayed for

radioactivity with a Nuclear Model D-47 gas flow counter

and standard scaling circuit.

For the TA experiments, female albino rats of the

Sprague-Dawley strain, weighing about 200 gm were used. TA

was injected subcutaneously as a 1% solution in isotonic 32 saline, in a dose of 5 mg/100 gm body weight. P -phos- 32 phate v/as injected intraperitoneally, 0.5 |ic of P /100 gm body weight, in isotonic saline neutralized with sodium hydroxide. 25 Three groups of rats were used in these experiments.

One group (Group A) was treated with TA for 3 days prior 32 to, and at the time of P injection. Another group

(Group B) received the first TA injection and P 32’ at the same time with daily TA injections thereafter, and a con- 32 trol group (Group C) received isotonic saline and P . At the time periods indicated in Figures 8 to 10, two animals from each group were killed by a blow on the head, the livers quickly removed and the nuclei isolated in 2.2 M sucrose according to the procedure of Chauveau et al.

(137). The nuclei were transferred to 15 ml conical centrifuge tubes and the washing and separation procedures, described above, were followed.

Results

Effects of Monovalent Cations

As shown in Table 1, isolated thymus nuclei are capable of utilizing a number of protein and nucleic acid precursors. The effect of sodium and potassium on the in­ corporation of these compounds into RNA, DNA, and protein is shown in Table 2. It is observed that the two amino acids tested, glycine and alanine, require the presence of sodium for incorporation into both protein and nucleic acid. In contrast, the nucleic acid precursors, adenine, uracil, phosphate, guanine, and AMP, do not exhibit this requirement. On the other hand, formate-carbon assimi- Table 1

Incorporation of the Carbon or Phosphorus of Various Labeled Compounds into Nuclear RNA, DNA, or Protein

C.P,M. incorporated into one mg RNA/ Compound per 300 ,000 C.P.M. of precursor DNA* RNA DNA PROTEIN 14 Glycxne-2-C 405 21 99 19 14 Alanine-1-C 765 51 324 15 14 Formate-C 3096 321 114 10 3 0 r thopho sph a te-P *? 435 45 6 10 1 4- Adenine-8-C 9618 153 60

, . 1 4 Guanme-4— C 11097 195 54 14 Uracil-2-C 120 0 Adenosine-5 1- monophosphate-8-C 5586 312 18

The complete system contained : 1. 0 ml o f nuclei in sucrose (86), 0.4 ml of sodium chloride 0.1 M. glucose solution (containing S.25 mg NaCl/ml), 0.5 ml 0.1 M sodium phosphate buffer (pH 7.1), and 0.1 ml of radioactive compound (containing approximately 300,000 cts/min). The mixtures were shaken at 38 C for 120 min. * Specific activity of RNA/Specific activity of DNA. Table 2

Effect of Replacement of Sodium by Potassium Ions on the Incorporation of Various Precursors into Nuclear RNA , DNA, and Protein

Activity in the presence of potassium Compound ion expressed as a per cent of the activity obtained with sodium ion

RNA DNA Protein 14 Glycine-2-c 24 20 22 14 Alanine-l-C 15 15 12 ] 4 Formate-C 84 111 55 3 2 Orthophosphate-P 104 97 105 Adenine-8-C1^ 123 115 Guanxne-4-C14 85 110 14 Jracil-2-C 123 Adenosine-51- ^ monophosphate-8-C 92 85

Conditions were the same as described with Table 1 except sodium ions were replaced by equal concentrations of potassium ions.

-j 28 lation into RNA or DNA is essentially the same, in the presence of sodium or potassium, but a sodium requirement

is shown for incorporation into protein.

The extent to which isolated thymus nuclei can utilize nucleic acid and protein precursors is illustrated in Table

1. Adenine and guanine, and to a lesser degree AMP, are most effectively utilized for RNA synthesis, while the up­ take of AMP into DNA is greater than is adenine or guanine.

Uracil, as expected, is not incorporated into DNA and its assimilation into RNA is low. As shown in the last column of Table 1, the compounds studied fall into at least 2 groups on the basis of their relative incorporations into

RNA. and DNA. Adenine and guanine are utilized most effi­ ciently for RNA synthesis, while glycine, alanine, AMP, formate, and phosphate are incorporated relatively well into both RNA and DNA.

The apparent requirement of sodium ions for the up­ take of glycine-carbon into nuclear RNA, DNA, and protein was further investigated by varying the concentrations of sodium and potassium ions. The results of a typical ex­ periment are shown in Figure 1. All three processes are inhibited essentially in the same manner by increasing potassium ion concentrations. That this is indeed an ex­ ample of a specific sodium requirement is further shown by the results presented in Table 3, where the ratio of the ions listed and sodium ions is equal to 1. It is observed Figure 1. Inhibition of the incorporation of incorporation the of Inhibition 1. Figure

% % Inhibition 60 20 0 4 50 30 potassium ions. potassium of quantities equivalent by placed of sodium by potassium ions. Complete Complete ions. potassium by sodium of al 1 xetsdu oswr re­ ionswere sodium except 1 Table DNA,RNA, nuclear into carbon glycine system was the same as described with with described as same the was system and protein by progressive replacement replacement progressive by andprotein DNA RNA mM Naj 29 Table 3

Effect of Partial Replacement of Sodium Ions by Various Monovalent Cations on Glycine Carbon Incorporation into RNA, DNA, and Protein

Ion Per cent of glycine carbon incorporation in the presence of sodium ions

RNA DNA Protein Potassium 71 64 77 Ammonium 62 78 75 Lithium 69 79 33 Rubidium 65 71 77 Caesium 70 70 73

Complete system contained: 1.0 ml of nuclear suspension, 0.1 ml of glycine-2-C (containing 650,000 cts/min), 0.2 ml 0.1 M glucose containing 6.25 mg NaCl/ml, 0.2 ml 0.1 M glucose containing a molar quantity of metal chloride equivalent to 6.25 mg NaCl/ml, and 0.5 ml 0.1 M sodium phosphate buffer (pH 6.96). The mixtures were shaken at 38°c for 120 min. 31 that all three processes are inhibited in the same manner by the partial replacement of sodium by another monovalent cation. That this sodium requirement is concerned in some way with amino acid utilization has already been indicated in Table 2.

32 Time Course of P Incorporation into RNA 32 As shown in Figure 2 the incorporation of P into the

RNA of isolated thymus nuclei proceeds at a linear rate for about 30 minutes before leveling off. This result is in agreement with a similiar finding of Allfrey and Mirsky

(87) who followed the incorporation of orotic acid and adenosine into nuclear RNA. It is interesting to note 32 that no time lag for the incorporation of P into RNA is observed. In contrast, Allfrey et _al. (86) have reported that a definite initial time lag of about 10 to 15 minutes is observed during the incorporation of labeled amino acids into nuclear protein.

Inhibitor Studies

In order to learn more about the interrelationships of the three processes under study, the effects of various known inhibitors of nucleic acid and protein syntheses were investigated.

Chloramphenicol has in the last few years become an important tool in the study of the possible relationship of protein and nucleic acid syntheses. In some systems 32

120

100

8 0

< o: 6 0

O' E

4 0

20

3 0 4 5 6 0 75 9 0 MINUTES

37 . . Figure 2. Time course of P incorporation into the RNA of isolated thymus nuclei. Complete system was the same as described with Table 1. 33

(138*139) chloramphenicol inhibits protein synthesis with­ out affecting nucleic acid synthesis* while in others (140-

142) both protein and nucleic acid syntheses are strongly inhibited. Allfrey et ad. (86) have reported a completely different effect in isolated thymus nuclei* where chlor­ amphenicol (at a concentration of 3.2 x 10 ^ M) failed to 14 inhibit the incorporation of C -amino acids into nuclear protein. Since this effect suggests that protein synthesis in nuclei might differ somehow from that in other systems* a more detailed study was initiated in which the effects of 14 chloramphenicol on both alanine-C incorporation into 32 protein and P -phosphate incorporation into the nucleic acids of isolated calf thymus nuclei were followed.

The effects of increasing concentrations of chloram- 14 phenicol on the incorporation of alanine-C into nuclear protein are shown in Figure 3. 7^.s chloramphenicol concen­ tration is increased above that used by Allfrey et _al. (86)* amino acid incorporation is progressively inhibited* and is completely inhibited at about 6.7 x 10 -3 M. Further- 32 more* although the rates of incorporation of P into RiSIA and DNA differ markedly* incorporation into both nucleic acids is inhibited in essentially the same manner as amino acid incorporation into protein. Chloramphenicol* there­ fore* apparently inhibits both protein and nucleic acid syntheses in isolated thymus nuclei in a manner similiar to that already observed in ascites tumor cells (140) and in Figure Figure

C.RM./mg NUCLEIC ACID 200 140 100 120 160 180 40 0300 60 80 20 . 3 361-' 3.6x10-*' 3.6x10-*' ) OA CNETAIN F CHLORAMPHENICOL CONCENTRATION OF MOLAR feto hoapeio n alanine-C on chloramphenicol of Effect into and (curve 1) protein into incorporation 2 3 p - e t a h p s o h p o h t r o A N R .X0 6.7x10-* * 6.7X10“ (curve ) 2 and A N D incorporation (curve . ) 3 1100 1000 700 1300 500 900 1200 600 100 800 200 400

4 1 34 35

isolated ribonucleoprotein particles (141,142).

Webster (142) has reported that both purine and 5-

mercaptopurine inhibit RNA and protein syntheses in iso­

lated ribonucleoprotein particles. The effects of these

purine antagonists on the utilization of glycine-C^ by

isolated thymus nuclei are illustrated in Figure 4. At

the concentrations employed in these experiments only gly- 14 cxne-C incorporation into RNA was consistently inhibited.

No inhibitory effect on glycine-C1^ incorporation into

either DNA or protein was observed. However, at higher _2 concentrations of purine (3.53 x 10 M) glycine incorpo­

ration into RNA, DNA, and protein was inhibited by 87%,

87%, and 81%, respectively. The low solubility of 6-

mercaptopurine prevented the use of this antagonist at higher concentrations.

The amino acid analogs, allylglycine and methionine

sulfoxide, inhibited all three processes, as shown in

Figure 5. In contrast to the results obtained when compa­

rable concentrations of purine and 5-mercaptopurine were

Uksed, amino acid incorporation into protein is inhibited

and glycine-carbon uptake into RNA is inhibited to a

greater extent.

The results of experiments where deoxyribonuclease

(DNAase) and ribonuclease (RNAase) were added to the in­

cubation medium are illustrated in Figure G . In these ex­ periments 51-57% of the RNA and 70-80% of the DNA were re- 36

% Activity 0 20 4 0 6 0 6 0 100 120 r - — i— T — r~ — I-

CONTROL

RNA

DNA 1 Purine 1 ( .68 x KT* M) PROTEIN |

1 RNA ' 1 1 6 - Mercoptopurine DNA | (.5 x I0-3 M )

PROTEIN 1 1

Figure 4„ Effect of purine and 5-mercaptopurine on the incorporation of glycine carbon into nuclear REA, DNA, and protein. Incubation system v/as the same as is given with Table 1 . % Activity

0,---- ,---- 2 0r---- ,---- 4, 0---- ,---- 61---- 0 1---- 81---- 0 1---- 1001---- r

CONTROL

RNA A lly I glycine DNA ( . 9 x I0“3 M)

PROTEIN

RNA

ONA Methionine Sulfoxide (. 5 x I0*3 M ) PROTEIN

Figure 5. Effect of amino acid analogs on the incorporation of glycine carbon into nuclear RNA, DNA, and protein. Incubation system was the same as described with Table 1. 38

% Activity 0 20 4 0 6 0 8 0 100 120 140 1 i i i i 1----- 1----- 1----- 1----- 1---- 1----- 1----- 1---- 1----- 1-

CONTROL

RNA

ONA RNAase (0.48 mg/ml)

PROTEIN

RNA DNAase DNA (0.17 mg/ml)

PROTEIN

Figure 6. Effects of ribonuclease and deoxyribo­ nuclease on the incorporation of glycine carbon into nuclear RNA, DNA, and protein. Incubation conditions were the same as previously, except the nuclei were incubated for 30 minutes with ribo­ nuclease or deoxyribonuclease before the addition of glycine-C14. moved by the action of the respective nucleases. In con­ firmation of the observations of Friedkin and Wood (94)

DNAase and not RNAase has an inhibitory effect on DNA synthesis. In addition;, it is also observed that RNAase treatment results in a substantial increase of the specif­ ic activities of both RNA and protein while DNAase treat­ ment has essentially the same marked inhibitory effect on both RNA and protein.

The surprising effect of RNAase treatment on the spec­ ific activities of RNA and protein appeared similiar to the effects reported by Ledoux (142) on Landschutz ascites cells. In contrast, Allfrey et al. (102) had previously observed that RNAase, in a final concentration of 0.063 mg/ml, had no effect on glycine-C^ incorporation into nuclear protein. A series of experiments were conducted in which the effect of varying concentrations of RNAase on the specific activities of nuclear RNA and protein was ob­ served. These results are shown in Figure 7. It is noted that above a concentration of 0.12 mg RNAase/ml the spec­ ific activities of the RNA and protein increase steadily.

To account for these increases in the specific activities of both RNA and protein the possibility could not be ex­ cluded that pretreatment of isolated nuclei with RNAase removed only "inactive'* RNA and protein. That this is in­ deed the case was made evident by the observation that the Figure 7. Effects of varying concentrations of concentrations varying of Effects 7. Figure A ctivity 160 140 120 100 80 - - 0

006 rti. nuainsse a the was system Incubation protein. glycine carbon into nuclear RNA and RNA nuclear into carbon glycine ribonuclease on the incorporation of incorporation the on ribonuclease ae s ecie ihFgr S. Figure with described assame

0.12 g RNAase/ml mg. 0.24 0.48 40 41 total activities of both the RNA and protein of RNAase treated nuclei were essentially the same as those of the controls.

The Effect of Thioacetamide on Nuclear RNA Metabolism in vivo

As discussed in the introduction, these experiments were undertaken to understand better and possibly to ex­ plain the rapid and specific increases in nuclear RNA of rat liver during thioacetamide (TA) treatment. Two sepa­ rate experiments were performed. In one, a study was made 32 of the P incorporation during the first 8 hours following 32 P injection (Fig. 8). The rats were fasted 24 hours 32 prior to the P injection. In a second experiment, with non-fasted rats, the synthesis and breakdown of nuclear

RNA was analyzed from samples taken 8.5 to 9S hours after 3 2 P injection (Figs. 9 and 10).

Figure 8 shows the specific activity/ DNA/RNA-time curves of the nuclear RNA of the three different groups of rats. Since TA treatment induces an increase in the nucle­ ar RNA, it was felt that a specific activity curve alone would not be as accurate an indication of the relative 32 total uptake of P as would the specific activity divided by the DNA/RNA ratio. TA administered in non-toxic levels for short periods of time has no effect on the DNA content per nucleus (130). The DNA/RNA ratio, therefore, gives an Figure 8. Specific activity/DNA/RNA-time curves activity/DNA/RNA-time Specific 8. Figure SPECIFIC ACTIVITY/ dna 0 0 5 0 0 4 200 300 100 0 ihT o 3 daysx^rior 3 for at TA and with to, 3 ax-C,p32 Group and time, same the received isotonic saline and p22. and saline isotonic received and injection TA first the received the time of P^2 injection. Group Group injection. P^2 of time the o ula N fGopA treated A, Group of RNA fornuclear 2 OR ATR 3 INJECTION P32 AFTER HOURS 3 4 5 - A 6

3,

7

8 42 43 accurate indication of the relative increase of nuclear

RNA per cell. It is observed in Figure 8 that the initial 32 rate of P incorporation is greater for Group A, while no appreciable difference is seen between Groups B and C. The 32 uptake of P proceeds at a linear rate over the first 4 32 hour period in Group B. The maximum P incorporation is reached in about 4 hours in Group A and the maxima for the two other groups are reached closer to 5 hours. 32 The total P incorporated into the RNA of Group A, at the maximum, is approximately double that of the controls 32 (Group C, Fig. 8). The rate of loss of P for Groups A and C is essentially the same. Group B incorporates and 32 loses P to a much greater extent than does the control during the first 8 hours. However, as shown in Figure 9, 32 the rate of P loss after 11 hours begins to proceed at a slower rate compared to that of the controls (Group C). In 32 contrast, the rate of P loss in Group A is greater after

11 hours.

During the initial 11 hours following a single inject­ ion of TA no change was noted in the DNA/RNA ratio of

Group B. At 24 hours following TA administration the

DNA/RNA ratio decreased from 4.5 to 3.8. At 48 hours, with continued treatment, this ratio was 3.4, and finally after 96 hours the ratio was 3.0. As can be seen from

Figure 10, there is a steady decrease in the DNA/RNA ratio

(increase in the RNA per nucleus) of Group B from 24-96 oltr Z H <|<

Figure 9. Specific activity/DNA/RNA-time curves activity/DNA/RNA-time Specific 9. Figure SPECIFIC A C T IV IT Y 200 0 0 3 0 0 5 0 0 4 100 hours and 8.5-96 hours represent two represent hours 8.5-96 and hours f h eprmn. aus o 1-5.5 for Values experiment. the of etta ru eevdte last the received A Group that cept ex­ 8 inFigure described as treated daily TA injections during the course the during injections TA daily A, Groups of RNA nuclear for eaae xeiet. h abscissa The experiments. separate administration and Group B received B Group and administration P^2 of time the at TA of injection s logarithmic. is _l 8.5 _ OR AFT P R TE F A HOURS 4 2 32 INJECTION B, and and C 8 4

44 6 9 iua 0 Efc fT nteDARArto of ratios DNA/RNA the on TA of Effect 10. Figura DNA/ RNA 4.5 .0 4 2.5 3.0 ecie nFgr 9. inFigure described rusA,.j n . odtos as Conditions .Bj andC. , A Groups 2 OR ATR P32 AFTER HOURS 4 55 . I 896 48 II 8.5 5.5 4 3 INJECTION 24 70

80 50 60 0 9 100

45 46 hours. After 4 days of daily treatment there is a 33% de­ crease in the DNA/RNA ratio (50% increase in REA/nucleus).

Also to be noted in Figure 10 is the initial decrease in the DNA/RNA ratio of Group A, followed by the steady in­ crease as the cells recover from the drug.

Effect of Thioacetamide on Isolated Thymus Nuclei

The effects of increasing concentrations of TA on the 14 incorporation of alanine-l-C into the protein of iso­ lated thymus nuclei are illustrated in Figure 11. The finding that only relatively high concentrations of TA af­ fected protein synthesis might indicate that a metabolic product of TA rather than TA itself is responsible for the enhancement of protein synthesis noted in experiments in vivo (130-132) . It was also found that at a concentration of 0.43 x 10 1 M, TA had no effect on the incorporation of 14 glycine-C into the RNA and DNA of isolated thymus nuclei. 47

2000

2 UJ V- o cc CL O' E 8 0 0 S CL d

4 0 0 -

4 x I0'4 4 x I0"3 4 x I0"2 4 x I0~ 1 4 MOLAR CONCENTRATION OF THIOACEl^MIDE

14 Figure 11. Effect of TA on alamne-c incorpo­ ration into the protein of isolated thymus nuclei. Incubation system was the same as is given with Table 1. 48

Discussion

The results presented in this study indicate that

isolated thymus nuclei are capable of utilizing the car­ bons of a diversity of known nucleic acid and protein pre­ cursors. The lack of a complete analysis of the RNA and

DNA purines and pyrimidines (i.e., separation into indi­ vidual bases and subsequent degradation studies to deter­ mine which carbons are labeled) prevents a rigorous inter­ pretation of these data. Also the possible presence of

intermediate "pools" which would tend to dilute out the

incorporation of radioactive precursors has not been

ascertained.

The finding that the carbon and/or carbons of simple precursors, such as formate and glycine, are incorporated

into the mixed nucleotides of DNA and RNA strongly suggests

that isolated nuclei are capable of the synthesis de novo of these polynucleotide bases. Furthermore, the incorpor­

ation of alanine carbon into both RNA and DNA is indicative

that the nucleus has the capacity to convert alanine to

aspartate and/or C02 . Aspartate could then be incorporated

into polynucleotide pyrimidine and CC>2 could be assimilated

into both polynucleotide pyrimidine and purine. The obser­ vation that the RNA/DNA incorporation ratios of glycine,

alanine, and AMP are essentially the same (Table 1) indi­ cates that the same metabolic route to polynucleotide is 49 common to these precursors. The considerably lower RNA/DNA incorporation ratio for formate may be due to formate participating in the methylation of uracil deoxyriboside

(144) to thymidine, a reaction which has been shown to occur at essentially the same rate, in vitro or in vivo

(82), while formate incorporation into purine is markedly decreased _in vitro (82,83) .

The considerably greater incorporation of the pre­ formed purines, adenine and guanine, into the nucleotides of RNA and DNA than the simple precursors, formate, glycine, and alanine, is in accord with the results of several workers using as experimental material ascites tumor cells

(82), rat liver (145-148), thymocytes (82,83), and ribo- nucleoprotein particles (141) . Furthermore, the difference in the ratio of incorporation of the preformed purines into

RNA and DNA compared to AMP would suggest that at least two biosynthetic pathways for nucleotide synthesis exist.

Also of interest in regard to AMP metabolism is its great­ er incorporation into DNA nucleotide than guanine or adenine. In contrast, adenine and guanine are incorporated to a greater extent into RNA nucleotide. These observations would appear to indicate that while the pathway of adenine to DNA polynucleotide involves the intermediate formation of AMP, its incorporation into RNA polynucleotide does not.

Since the mixed RNA nucleotides were assayed in these experiments and because adenine is known to be converted to RNA guanine (88,149), the possibility cannot be dismissed

that only a portion of the incorporation of adenine into

RNA is present as RNA adenine. If only 50% of adenine were converted to RNA guanine, then the incorporation of adenine

into RNA adenine would be a value which is the same as AMP

incorporation into RNA. This possibility is however not

tenable for the following reasons: (1) AMP may also be partially converted to the guanine derivative and (2) the

recent work of Logan (88) indicates that the conversion of

adenine to RNA guanine occurs to a lesser degree than that which would be of significance here. In studying the in- 14 corporation of adenine-8-C into RNA of isolated thymus nuclei, Logan reports the following data: in two hours incubation (with the same materials and conditions used in 14 this study) adenine-8-C incorporation into RNA I and RNA

II was 38,000 and 8,000 c.p.m./(imole adenine while the in- i 4 corporation of adenine-8-C ' into the guanine of RNA I and

RNA II, was 6,000 and 2,000 c,p.m./(imole guanine. The percent conversion of adenine to guanine was therefore approximately 15% for RNA II and 25% for RNA I. Allfrey and Mirsky (87) have reported that in isolated thymus nu­ clei there is approximately 1.6 times as much RNA I as RNA

II. Applying this value to Logan's data would indicate that only about 22% of the adenine utilized for RNA nucleo­ tide biosynthesis was converted to RNA guanine. If this 51 correction is made in Table 1, then the value for the adenine which is converted to RNA adenine would be about

7700-a value which is still too high for considering AMP as an intermediate in the incorporation of preformed adenine into RNA.

The finding that adenine is a much better precursor of nucleic acids than AMP has its parallel in studies in vivo. Lowry et al. (150) have demonstrated that labeled adenosine is incorporated about only one-half as well as adenine into the RNA and DNA of rats. Experiments using rats (151), and tissue slices and cell suspensions (152) have indicated that when either 3 ‘- or 5'-nucleoside mono­ phosphates, labeled with P ^ , are employed as precursors, 32 the P is separated from the nucleoside and appears ran­ domly distributed among ail four nucleotides obtained from labeled RNA. These results have been explained by the impermeability of animal cells to mononucleotides, which are presumably cleaved at the cell membrane (152) . That

AMP can be a precursor of cytoplasmic RNA has been shown 32 by Heidelberger et al. (153) by following AMP incorpora­ tion into the cytoplasmic fraction of rat liver homogenates.

They found that the incubated nucleotide was incorporated into RNA without loss or randomization of its phosphorus, thus showing that 5 1-nucleotides are intermediates in RNA biosynthesis.

The possibility that the AMP incorporation into nu- 52 clear RNA is lower than adenine, because of a nuclear mem­ brane block, does not appear tenable because of the find­ ing that AMP is incorporated better into DNA than is aden­ ine. AMP transport into the nucleus is therefore appar­ ently not blocked to any appreciable extent.

In an attempt to explain the differential incorpora­ tions of adenine and AMP into polynucleotide the following speculative scheme is proposed.

adenine-* dR-adenosine-) dR-AMP—) dR-ADP—> dR-ATP------^ DNA

adenosine ^ AMP ■> ADP } ATP

glycine, CO formate

The key to this scheme is the concept of a "bound"

AMP. The suggestion that a "bound" AMP exists in nuclei was first proposed by osawa et al. (125) to explain the finding that only intranuclear AMP was converted to ATP in isolated thymus nuclei. These workers found that addition of over twice as much AMP as was present in isolated nuclei along with an appropriate amount of inorganic phosphate re­ sulted in no net increase of ADP or ATP as compared to the controls to which no AMP had been added. Furthermore, the 53 added AMP could be recovered quantitatively.

Because of the known relatively fast turnover of nu­ clear RNA in comparison to that of DNA, an equally fast conversion of adenine to "bound" adenosine or adenosine derivatives would be expected to occur in the above specu­ lative scheme. This scheme also presupposes that when AMP is added, in order for it to be incorporated into RNA, it would have to be converted first to adenine via adenosine, a series of reactions which are known to occur but whose equilibrium points favor the formation of the more highly substituted derivatives. Therefore, relatively small amounts of AMP v/ould be expected to be converted to aden­ osine or adenine and that which does may be diluted ex­ tensively by an existing pool. The conversion of AMP to

DNA nucleotide would most probably occur by conversion of the ribosyl moiety to the deoxyribosyl moiety at the nu­ cleotide level. A more direct course of AMP incorporation into DNA therefore exists than that for adenine. This scheme would also illustrate why glycine and alanine are incorporated into RNA and DNA in a manner similiar to AMP.

The nature of the material to which AMP might be bound is very questionable. One is very tempted to suggest DNA or DNA-protein because of the central role that DNA appears to play in both RNA and DNA synthesis in isolated nuclei.

The studies reported here on the inhibition of DNA and RNA syntheses are of interest in this connection. In every case examined, inhibition of RNA metabolism is not neces­ sarily accompanied by inhibition of DNA or protein metab­ olism, while inhibition of DNA metabolism- is always accom­ panied by an inhibition of protein and RNA metabolism.

Likewise, removal of RNA with ribonuclease fails to inhib­ it precursor incorporation into DNA or protein, but DNA re­ moval results in an inhibition of incorporation into both protein and RNA. If, as has been suggested (86), DNA is necessary for nuclear protein synthesis, then the same kind of evidence, obtained in this study, could be interpreted as indicating that DNA is also necessary for nuclear RNA synthesis. This, in turn, suggests the possibility that

DNA is formed from nucleotides by a fairly independent process while RNA is formed by some mechanism involving

DNA. If the constituent ribonucleotides of RNA were ar­ ranged on the DNA or DNA-protein at a distance of 1 nucleo­ tide from each other, and if amino acid-nucleoside diphos­ phate complexes were formed with them, then protein synthe­ sis and RNA synthesis could proceed in the manner suggested by Webster (142), where the breaking of the amino acid- diphosphate bond would allow protein synthesis to occur while the nucleoside diphosphates can polymerize along the

DNA template (through the mediation of polynucleotide phosphorylase) forming RNA. In this way, DNA can serve as the directing force in synthesizing a specific RNA and protein-, 55

The finding in this study, that removal of DNA by

DNAase-treatment markedly inhibits RNA and protein synthe­ sis (Fig. 6 ), is in agreement with the proposed scheme.

Also, RNAase should have no effect on DNA, RNA, and protein synthesis and experimentally this was also found (Fig. 5).

According to this proposed scheme, protein synthesis would not occur unless nucleoside diphosphates were first

"bound" to the DNA or DNA-protein. If the reverse situa­ tion existed, whereby nucleoside diphosphates complexed with amino acids which were bound in some way to DNA or

DNA-protein, then the effects of DNAase and RNAase would still be the same and furthermore the effects of the amino acid and purine analogues observed in this study could be more clearly explained. Protein synthesis could then continue in the absence of RNA synthesis, a phenomena ob­ served in the presence of purine and S-mercaptopurine (Fig.

4) . Allfrey e_t al. (86) have observed a similiar effect of DRB on isolated thymus nuclei. The amino acid ana­ logues, allylglycine and methionine sulfoxide, inhibited

RNA synthesis to a greater extent than protein synthesis while having only a slight effect on DNA synthesis (Fig. 5).

The utilization of preformed guanine by isolated nu­ clei has not been hitherto reported on. In mammalian tissue, guanine is incorporated in very small amounts into nucleic acid guanine (154) and is converted to RNA adenine in only trace amounts (145). In contrast, this study indi­ 56 cates that in isolated nuclei the incorporation of guanine is as extensive as that of adenine and follows a similiar pathway to polynucleotide as indicated by the RNA/DNA ra­ tios (Table 1). The possibility that guanine or a deriv­ ative is converted to adenine or its derivative before.in­ corporation appears questionable since, of known biological matter, only a few bacterial species can convert guanine into polynucleotide adenine to an extent which would be of significance here (155-151) .

The extreme dependence of isolated thymus nuclei on the presence of sodium ions for amino acid incorporation into protein is in sharp contrast with the results of oth­ er workers (152-165) who have found a potassium requirement using a variety of biological materials. The inhibition of glycine and alanine carbon incorporation into polynucleo­ tide in the absence of sodium in the incubation medium is also contrary to the known potassium ion requirements for at least two steps in the synthesis of purines de novo; the formation of formylglycinamid.ine ribotide from formyl- glycinamide ribotide and the formation of 5-formamido-4- imidazole-carboxamide ribotide from 5-amino-4-imidazole- carboxamide ribotide (156) . The finding that formate carbon incorporation into polynucleotide was only slightly inhibited by the absence of sodium in the medium while being substantially inhibited into protein, suggests very strongly that sodium is involved in some way only with amino acid metabolism in isolated nuclei. This view is further strengthened by the finding that AMP, guanine, adenine, phosphate, and uracil incorporation into poly­ nucleotide was essentially unaffected by the absence or presence of sodium in the medium. Why sodium should be . required for some aspect of amino acid metabolism (trans­ port?) is not very clear. Griswold and Pace (167) have reported that liver cell nuclei contain about 13% of the sodium and potassium of the liver cell. If essentially the same relationship should also be the case in thymus nuclei, then the additional requirement for sodium becomes even more an unknown requirement.

The small incorporation of uracil into nuclear RNA is in agreement with experiments in vivo using rats (168,159) .

On the other hand, Webster (14-1) has found that pea ribo- nucleoprotein particles incorporate uracil extensively in­ to ribopolynucleotide. In contrast, Hecht et al. (93) have shown that UTP and UDP are incorporated into the sol­ uble RNA of ascites cells to a much smaller extent than the adenine and cytosine derivatives. 32 The data presented on the incorporation of P into the nuclear RNA of thioacetamide treated rat liver indi­ cates that TA induces an increase in the synthesis of nu­ clear RNA. This is shown by both the greater initial rate of P 32 uptake and the total p 32 incorporated (Group A,

Fig. 8). The initial effect of TA results in an enhance­ ment of both nuclear RNA synthesis and breakdown (Group B, 32 Pig. 8). The rate of loss of P is lower than control

levels 11 hours after the initiation of TA treatment (Group

B, Fig. 9). This can be attributed either to a decrease in

RNA degradation or more likely to a reutilization of de­

graded RNA components accompanied by increased RNA synthe­

sis. Morphological changes are first noted in the nuclei

20-24 hours after drug treatment suggesting that the total

RNA content of the nuclei is, in effect, the same as the

controls during the early hours in spite of the observed

increase in RNA synthesis. As can be seen from Figure 10,

Group B, the DNA/RNA ratio is markedly decreased at 24

hours and continues to decrease with continued treatment.

The net nuclear RNA metabolism of liver cells sub­

jected to TA treatment for three days (Group A), differs

in many respects from that of liver cells after a single

injection (Group B). There is a rapid incorporation of 32 P into the nuclear RNA of Group A and a steady decrease

in the DNA/RNA ratio (increase in nuclear RNA) during an 32 8.5 hour period following TA and P administration. As

noted above, during this same time period, no net change

occurs in the nuclear RNA in Group B. Also of interest

with regard to Group A is the rapidity with which the cell

appears to recover when TA is stopped. Eleven hours after 32 the final TA injection, the rate of P loss begins to in­

crease, and parallels the decrease in nuclear RNA (increase in the DNA/RNA ratio). This contrasts with the continued increase in nuclear RNA of Group B receiving daily TA treatment. It is conceivable that TA (or a metabolite) directly enhances nuclear RNA synthesis whereas the in­ creased RNA degradation reflects an attempt of the cell to cope with the increased levels of RNA and prevent its accumulation. The rapid recovery upon withdrawal of drug treatment supports this postulate.

A more complete explanation of the action of TA on the metabolism of the liver cell must await further experi­ mentation. 60

Summary

Isolated calf thymus nuclei incorporate the carbons of glycine, alanine, formate, adenine, guanine ■, ' and adenosine-5'-phosphate, as ■well as orthophosphate,’-, into’ both DNA and RNA.- - Adenine is ’incorporated' more- readily ' ■' than adenosine-5 1-phosphate into- RNA,' while-.afle-nbeine-S.'- phosphate is incorporated into DHA-.more re-adi-ly than ad­ enine. Incorporation of glycine or alanine carbons, into both DNA and RNA requires, sodium ions, and the sodium, can­ not be replaced by potassium, ammonium, -lithiUm,-■ rubidiumy or caesium ions. incorporation of all precursors except amino acids, however, exhibits no.sodium requirement. Re­ moval. of DNA with deoxyribonuclease results in strong in­ hibitions of precursor incorporation into, both RNA and'.pro^ tein, but removal of RNA with ribonuclease does not inhibit. incorporation into either DNA or -protein. 32 •. ■ ■ ■ The P . uptake i ^ the nuclear-'ribonucleic acid -fr.ac- • tion of rat liver cells was investigated i n .animals treat-'- ed with thioacetamide (TA) ,- a drug reported to induce in­ creased nuclear RNA and protein'. The. data* presented i’n«. - dicate that TA induces an increase in the synthesis of 32 nuclear RNA as shown by the greater initial rate of P 32 incorporated and the total P uptake. The rate of break­ down of nuclear RNA is increased during the initial 8 hour period following a single injection of TA. Within 24- 61 hours and with continued daily treatment the rate of loss 32 of P is below control levels, while the nuclear RNA synthesis continues at an increased rate. Rats treated for 96 hours (4 daily injections) showed a 50% increase in the nuclear RNA/cell as determined by the DNA/RNA ratios*.

‘The data presented strongly suggest that the observed in­ crease,''in nuclear 'RNA . is . due to an enhancement .of'RNA “ ; synthes'is;... ■ • ’ ‘ • * ' . 62

Bibliography

1. V. G. Allfrey, A. S. Mirsky, and H. Stern, Advances in

Enzymol. 16, 411 (1955).

2. A. L. Bounce, in "The Nucleic Acids" (Chargaff and

Davidson, • eds .) , Vol., II, p. 93. Academic Press, New

York, 1955.. • " •

3. G.- B. Brown and _P-._ M. Roll, -.in "The -Nucleic Acids"

. • - (Char.gaff arid Davidson, eds.)', Vol. II, p. -341..

Academic Press, ..New York, 1955.

• 4 R;. M .: ,S .■ Smellie, in "The Nucleic Acids" (Chargaff and

./Davidson, eds:.), Vol. II, p. 393. Academic Press,. New

York, 1955. • •

5. G-. Siebert. arid. R . r M-. -S-. Smellie, Intern. Rev. Cytol.

• 6, 383 (1957).

6. D. Mazi’a, in "Enzymes: Units of .Biological Structure

. and. Function." (Gaebler, ed.) , p'. 261. . Academic -Press,

New York,- 1956. ’ , . ..

7. D-.- Mazia/ in "Modern Trends ‘in .'.Physiology arid Bio-

•. .chemistry" '• (Baron, ed.-)',. Academic Press, New-York, 1952.

8. A. Marshak-,. J. Cellular Comp. Physiol. 3_2,-381 (1948)1

9. A. Bergstrand, N. A. Eliasson, B. Norberg, P. Relchard,

. and H. von Ubisch, Cold Spring Harbor Symp. Quant.

Biol. 13, 22 (1948).

10. R. Jeener, Nature 163, 837 (1949) .

11. R. Jeener and D. Szafarz, Arch. Biochem. 23, 54 (1950). 12. C. P. Barnum and R. a. Huseby, Arch. Biochem. .29, 7

(1950) .

13. C. P. Barnum, R. A. Huseby, and H. vermund, Cancer

Research L3, 330 (1953) .

14. W. M. MeIndoe and J. N. Davidson, Brit. J. Cancer S.,

200 (1952) .

15. E. P. Anderson and S. E. G. Aquist, J. Biol. Chem.

202. 513 (1953) .

15. R. M. S. Smellie, W. M. Mclndoe, and J. N . Davidson,

.Biochem, et Biophys. Acta 13., 559 (1953).

17. R. M. 'S.- Smellie, W. M. Mclndoe, R. Logan, J. N.

.Davidson, -and I. Dawson, Biochem. J. 54, 280 (1953).

18. J. R.' Fresco and A. Marha'k, J. Biol. Chem. 205, 535

(1953) .

19.' R. B. Hurlbert and V. R. Potter, J. Biol. Chem. 209.

• 1 (1954) .

20. A. Marshak and F. Calvet, J. Cell. Comp. Physiol. 34,

451 (1949).

21. J. N. Davidson and R. M. S. Smellie, Biochem. J. 52,

594, 599 (1952).

22^ D. Elson, L. W. Trent, and E. Chargaff, Biochem. et

Biophys. Acta 17., 362 (1955) .

23. G. W. Crosbie, R. M. S. Smellie, and J. N. Davidson,

Biochem. J. _54, 287 (1958) .

24. S. Osaw;i, K. Takata, and Y. Hotta, Biochem. et Biophys.

Acta 28., 271 (1958) . 64

25. Y. Hotta and S. Osawa, Biochim. et Biophys. Acta 28,

642 (1958).

26. R. Logan and J. N. Davidson, Biochim. et Biophys.

Acta 24, 196 (1957).

27. N. Linet and J. Brachet, Biochim. et Biophys. Acta _7,

S07 (1951).

28. T. W. James, Biochim. et Biophys. Acta _15, 367 (1954).

29. L. Goldstein and W. Plaut, Proc. Natl. Acad. Sci.

(U.S .) 41,. 874 (1955) .

30. W. Plaut and R. C. Rustad, Nature 177, 89 (1956).

31. J. Brachet and D. Szafarz, Biochim. et Biophys. Acta

•12-, 580 (1953) .

32 . J. Brachet, H. Chantrenne, and F. Vanderhaeghe,

Biochim. et Biophys. Acta 18, 544 (1955).

33. H. Stich and W. Plaut, J. Biophys. Biochem. Cytology

4, 119 (1958) .

34. L . S . Lockingen and A. G. DeBusk, Proc. Natl. Acad.

Sci. (U.S.) 41, 925 (1955).

35. H. Fraenkel-Conrat, B. Singer, and R. C. Williams,

Biochim. et Biophys. Acta _25, 87 (1957) .

36. J. S. Colter, H. H. Bird, A. W. Moyer, and R. A.

Brown, Virology 4, 522 (1957).

37 . G. Hevesy, “Radioactive Indicators". Interscience,

New York, 1948.

38 . A. M. Brues, M. M. Tracy, and W. E. Cohn, J. Biol.

Chem. 173, 233 (1948). 65

39. E. Andreasen and J. ottesen, Acta Physiol. Scand. _5,

2.37 (1945) .

40. E. E . Osgood, H. Tivey, K. B. Davidson, A . J. Seaman,

and J. G. Li, Cancer 5_, 331 (1952) .

41. A. Bendich, Exptl. Cell Research Suopl. 2, 181 (1952) .

42. C. P. Sarnum, C. D, Jardetzky, and F. Holberg, Texas

Rep. Biol, and Med. 15, 134 (1957).

43. L. I. Hecht and v. R. Potter, Cancer Research _16, 988

(1956) .

44. Y. Takagi, L. I. Hecht, and V. R. Potter, Cancer

Research 1S_, 994 (1956) .

45. A. Bendich, P. J. Russell, and G. B. Brown, J. Biol.

Chem. 203, 305 (1953).

45. A. Bendich in "Essays in Biochemistry" (Graff, ed.),

p. 14. John Wiley and Sons, Inc. Hew York, 1956.

47. A. Bendich, H. B. Pahl, G. C. Korngold, H. S.

Rosenkranz, and J. R. Fresco, J. Am. Chem. Soc. 80,

3949 (1958) .

48. G. L. Brown and M. Watson, Mature 172, 289 (1953).

49. E. Chargaff, C. F. Crompton, and R. Lipshitz, Mature

172, 32 (1954).

50. J. 'A. Lucy and J. A. V. Butler, Mature 174, 32 (1954) .

51. A. Bendich, J. R. Fresco, H. S. Rosenkranz, and S. M.

Beiser, J. Am. Chem. Soc. T7, 3671 (1955).

52. A. Bendich, H. 3. Pahl, and S. M. Beiser, Cold Spring

Harbor Syrup. Quant. Biol. 2JL, 31 (1956). 53. R. Bachmann and E . Harbers, Biochim. st Biophys. Acta

16, 604 (1955).

54. T. Caspersson, Naturwissenschaften 28_, 33 (1941) .

55 . T. Caspersson, Symposia Soc. Exptl. Biol. JL, 127

(1947) .

55 . T. Caspersson, “Cell Growth and Cell Function1,

Norton, New York, 1950.

57 . to. M. Daly, v. G. Allfrey, and A. E. Mirs'ky, J. Gen.

Physiol. _36, 173 (1952).

50 . R. to. S. Smellie, W. to. Mclndoe, R. Logan, J. N .

Davidson, and I. Dawson, Biochem. J. 54, 280 (1953).

59. R. to. S. Smellie, W. to. Mclndoe, R. Logan, J. N.

Davidson, Biochim. et Biophys. Acta _11, 599 (1953).

50. H. Borsook, C. L. Deasv, A. J. Haagen-Smit, G.

Keighley, and P. H. Lowy, Federation Proc. 9_, 154

(1950).

51. T. Iiultin, Exptl. Cell Research 376, 599 (1950).

52 . E. P. Tyner, C. H. Heidelberger, and G. • ILSP clCJS j

Federation Proc. 11., 300 (1952).

S3. E. P. Tyner, C. H. Heidelberger, and G. A. LePage,

Cancer Research 1_2, 158 (1952) .

64. E. B. Keller, Federation Proc. _10, 105 (1951) .

65. N. D. Lee, J. T. Anderson, R. Miller, and R. H.

Williams, J. Biol. Chem. 192, 733 (1951).

66 . G. C. Webster, Ann. Rev. Plant Physiol. 5, 43 (1955).

67 . A. Ficq, Experientia _9, 377 (1953) . 67

68. A. Ficq and M. Erreraj Biochim. et Biophys. Acta 16,

45 (1955).

69. I. M. London, D. Shemin, and D. Rittehberg, J. Biol.

Chem. 183, 749 (1950).

70. H. Borsook, C. L. Deasy, A. J. Haagen-Smit, G.

Keighley, and P. H. Lowy, Federation Proc. 1Q_, 18

(1951) .

71. H. Borsook, G. L. Deasy, A. J. Haagen-Smit, G.

Keighley, and P. H. Lowy, J. Biol. Chem. 196, 669

(1952) .

72. B. W. Holloway and S. H. Ripley, J. Biol. Chem. 196,

695 (1952) .

73. J. Hammerling, Wilkelm Roux, Arch. Entwicklungsmech.

Organ. 13_1, 1 (1934) .

74. F. Vanderhaeghe, A.rch. intern, physiol. 6J3, 190 (1952).

75. J. Brachet and H. Chantrenne, Nature 168, 950 (1951) .

76. J. Brachet and H. Chantrenne, Arch, intern, physiol.

60, 547 (1952) .

77. J. Brachet and J. Brygier, Arch, intern, physiol. 61,

248 (1953) .

78. J. Brachet and H. Chantrenne, Arch, intern, physiol.

61, 246 (1953) .

79. H. Chantrenne, J. Brachet, and O'. Brygier, Arch,

intern, physiol. 6JL, 419 (1953).

80. J. N. Davidson, Proc. 3rd Intl. Congr. Biochem.,

Brussels, p. 353, 1955. 81. R. M. S. Smellie, R. Y. Thomson, R. Goutier, and J. N.

Davidson, Biochim. et Biophys. Acta _22, 585 (1956).

82. R. M. S. Smellie, R. Y. Thomson, and J. N . Davidson,

Biochim. et Biophys. Acta 2j), 59 (1958).

83. R. Y. Thomson, R. M. S. Smellie, and J. N. Davidson,

Biochim. et Biophys. Acta 2J9, 308 (1958).

84. L. I. Hecht and V. R. Potter, Cancer Research _18, 186

(1958).

85. G. Siebert, K. Lang, S. Lucius, and G. Rossmuller,

Biochem. Z. 324, 311 (1953).

86. V. G. Allfrey, A. E. Mirsky, and S. Osawa, J. Gen.

Physiol. 40, 451 (1957).

87. V. G. Allfrey and A. E. Mirsky, Proc. Natl. Acad. Sci.

(U.S.) 43, 821 (1957).

88. R. Logan, Biochim. et Biophys. Acta _26, 227 (1957).

89. R. Logan and R. m . S. Smellie, Biochim. et Biophys.

Acta 2JL, 92 (1955) .

90. R. J. Hilmoe and L. A. Heppel, J. Am. Chem. Soc. 79,

4810 (1957) .

91. S. Ochoa and L. A. Heppel in "Chemical Basis of

Heredity" (McElroy and Glass, eds.), p. 200. Johns

Hopkins Press, Baltimore, .1957 .

92. E. S. Canellakis, Biochim. et Biophys. Acta 2_5, 21.7

(1957).

93. L. I. Hecht, P. C. Zamecnik, M. L. Stephenson, and J.

F. Scott, J. Biol. Chem. 233, 954 (1958). 69

94. M. Friedkin and H. Wood, IV, J. Biol. Chem. 220, 639

(1956).

95. T. P. Wang, H. Z. Sable, and J. 0. Lampen, J. Biol.

Chem. 184, 17 (1950).

96. W. S. MacNutt, Biochem. J. _50, 384 (1951-52).

97. F. Weygand, A. Wacker, and H. Dellweg, Z. Naturforsch.

5b, 130 (1951).

98. W. Andersen, C. A. Dekker, and A. R. Todd, J. Chem.

Soc. 2721 (1952) .

99. M. Friedkin and D. Roberts, J. Biol. Chem. 220, 653

(1955) .

100. P. Reichard, Acta chem. Scand. 9, 1275 (1955).

101. A. Bendich, J. R. Fresco, I-I. S. Rosenkranz, and S. M.

Beiser, J. Am. Chem. Soc. _77, 3671 (1955).

102. V. G. Allfrey, A. E. Mirsky, and S. Osawa, Nature

175, 1042 (1955) . o 103. A. Kornberg, I. R. Lehman, and E. S. Simms, o Federation proc. _15, 291 (1955).

104. A. Kornberg, I. R. Lehman, M. J. Bessman, and E . S.

Simms, Biochim. et Biophys. Acta _2l, 197 (1956).

105. M. J. Bessman, I. R. Lehman, E . S. Simms, and A.

Kornberg, Federation Proc. _1S, 153 (1957).

106. I. R. Lehman, M. Bessman, E. S. Simms, and A.

Kornberg, J. Biol. Chem. 233, 153 (1958).

107. M. Bessman, I. R. Lehman, E. S. Simms, and A.

Kornberg, J. Biol. Chem. 233, 171 (195C) . 70

108. F. J. Bollum and v. R. Potter* J. Am. Chem. Soc. 79*

3503 (1957) .

109. F. J. Bollum and V. R. Potter* J. Biol. Chem. 233,

478 (1958) .

110. R. Mantsavinos and E. S. Canellakis* Biochim. et

Biophys. Acta 257* 561 (1958).

Ill. M. Sekiguchi and A. Sibatani* Biochim. et Biophys.

Acta 28* 455 (1958) .

112 . K. Lang* H. Lang* G. Siebert* and S. Lucius* Biochem.

Z. 324* 217 (1953).

113. V. G. Allfrey* Proc. Natl. Acad. Sci. (U.S.) 40* 881

(1954).

114. P. Siekevitz* J. Biol. Chem. 19 5, 549 (1952).

115 . M. Beljanski* Biochim. et Biophys. Acta 15 * 425

(1354).

13. o . E. F. Gale* Advances in Protein Chem. 8_* 285 (1953) .

117 . A. B. Pardee* proc. Natl. Acad. Sci. (U.S.) 40* 253

(1954).

118 . E. H. Greaser* Nature 176, 555 (1955).

119. G. C. Webster and M. P. Johnson* J. Biol. Chem. 217*

G41 (1955) .

120 . P. C. Zamecnik and E. B. Keller* J. Biol. Chem. 209,

337 (1954) .

121. G. C. Webster in "Chemical Basis of Heredity"

(McElroy and Glass* eds.) p. 2 6 8 . Johns Hopkins

Press* Baltimore* 1957. 71 122. v. G. Allfrey, M. M. Daly, and A. E. Mirsky, J. Gen.

Physiol. 37, 157 (1953).

123. V. G. Allfrey, A. E. Mirsky, and S. Osawa, Nature

175, 1042 (1955) .

124. V. Allfrey, A. E. Mirsky, and S. Osawa in “Chemical

Basis of Heredity” (McElroy and Glass, eds.) p. 200.

Johns Hopkins Press, Baltimore, 1957.

125. S. Osawa, V. G. Allfrey, and A. E. Mirsky, J. Gen.

Physiol. 40, 431 (1957).

125. V. G. Allfrey and A. E. Mirsky, Proc. Natl. Acad.

Sci. (U.S.) 43, 589 (1957).

127. H. Chantrenne, Ann. Rev. Biochem. 27, 35 (1953).

123. L. A. Heppel and j. c. Rabinowits, .Ann. Rev.

Biochem. 21_, 513 (1958) .

129. V . G. Allfrey and A. E. Mirsky, Proc. Natl. Acad.

Sci. (U.S.) 44, 981 (195S) .

130. A. K. Laird, Arch. Biochem. Biophys. 45, 119 (1953).

131. R. G. Kleinfeld, Cancer Research 17, 954 (1957).

132. L. J. Rather, Bull. Johns Hopkins Hosp. SO, 33 (1951)

133. G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 131,

33 (1945).

134. W. J. Kleinschmidt and J. A. Manthey, Arch. Biochem.

Biophys. 73., 52 (195S) .

135. W. E. Cohn and E. Volkin, Nature 137, 483 (1951) .

135. A. G. Cornell, C. J. Bardawill, and M. M. David, j.

Biol. Chem. 117, 751 (1949). 72

137. J. CliauveaUj Y. Moule and Ch. Rouiller, Exp. Cell

Research _11, 317 (1955) ,

138. E . F. Gale and J. p. Polices, Biochem. J. 5_3, 493

(1953).

139. C. 'L. Wissernan, Jr., J. E. Smadel, F. E. Hahn, and

H. E. Hopps, J. Bacteriol. _57, 652 (1954).

140. G. A. LePage, Proc. Soc. Exptl. Biol. Med. 0J3 , 724

(1953).

141. G. C. Webster, Arch. Biochem. Biophys. _58, 403 (1957).

142. G. C. Webster, Acta histochernica 4, 9 (1957).

143. L. Ledoux, Biochim. et Biophys. Acta _20, 369 (1955).

144. R. L. Blakley, Biochim. et Biophys. Acta 2_4, 224

(1957) .

145. S. S. Furst and G. B. Brown, J. Biol. Chem. 191, 239

(1951).

14-5. R. Abrams, Arch. Biochem. Biophys. 3_3, 435 (1951).

147. D. A. Goldtbwait and A. Bendich, J. Biol. Chem. 196,

841 (1952).

148. S. S. Furst, P. M. Roll, and G. B. Brown, J. Biol.

Chem. 183, 251 (1950) .

149. G . B. Brown, P. M. Roll, A. A. Plentl, and L. F.

Cavalieri, J. Biol. Chem. 17 2, 459 (1948).

150. B. A. Lowry, J. Davoll, and G. B. Brown, J. Biol.

Chem. 197. 591 (1952) . V 151. P. M. Roll, H. Weinfeld, and G. B. Brown, Biochim.

et Biophys. Acta 13*, 141 (1954) . 73

152 . K. C. Leibman and C. Heidelberger, J. Biol. Chem.

216, 823 (1955) .

153. C. Heidelberger, E. Harbers, K. C. Leibman, Y.

Tahagi, and V. R. Potter, Biochim. et Biophys. Acta

_20, 445 (1955) .

154. M. E. Balis, D. H. Marrian, and g . B. Brown, J. Am.

Chem. Soc. 73, 3319 (1951°).

155. M. E. Balis, G. B. Brown, G.°B. Elion, G. H.

Hitchings, and H. VanderWerff, J. Biol. Chem. 188,

217 (1951).

155 . M. E. Balis, D. H. Levin, G. B. Brown, G. B. Elion,

H. VanderWerff, and G. H. Hitchings, J. Biol. Chem.

o 195, 729 (1952) . o 157 . G. B. Elion, H. VanderWerff, G. H. Hitchings, M. E.

Balis, D. H. Levin, and G. B. Brown, J. Biol. Chem.

200, 7 (1953) .

158 . G. B. Elion, S. Singer, G. H. Hitchings, M. E. Balis,

and G. B. Brown, J. Biol. Chem. 202, 647 (1953).

159. M. Flavin and S. Graff, J. Biol. Chem. 191, 55 (1951) .

150. M. Flavin and S. Graff, J. Biol. Chem. 195, 485 (1951).

151. F. Weygand, A. Wacher, and H. Dellweg, 3. Haturforsch.

7b, 156 (1952) .

152 . F. C. Steward and C. Preston, Plant Physiol. _16, 85

(1941).

153. P. R. Cannon, L. E. Frazier, and R. H. Hughes, Metab­

olism, Clin, and Exptl. _1, 49 (1952). 74

164. O. E. Landman and S. Spiegelman, Proc. Natl. Acad.

Sci. (U.S.) 41, 638 (1955).

165. G. C. Webster, Biochim. et Biophys. Acta _20, 565

(1955) .

166. J. M. Buchanan, J. G. Flaks, S. G. Hartman, B.

Levenberg, L. N. Lukens, and L. Warren, Ciba o Foundation Symposium on the Chemistry and Biology

of Purines, p. 233, Little, Brown Co., Boston, 1957.

167. R. L. Griswold and N . Pace, Exp. Cell Research 11,

352 (1956) .

168. A. A. Plentl and R. Schoenheimer, J. Biol. Chem. o 153. 203 (1944).

169. R. J. Rutraan, A. Cantarow, K. E. Paschkiss, and B.

Allanoff, Science 117, 282 (1953) .

o a,utobiQ£traphv

I, Theodore Ronald Breitm^n, was born In Brooklyn,

Mew York, February 25, 1931* 3; received cry secondary school education in the public schools of Brooklyn, Mew

York, and my undergraduate training at the City College of New York, which granted me trhe Bachelor of Science degree in 1953. From The Ohio State University, 1 re­ ceived the Master of Science d^gree 1953, While in residence there, I was an Assistant in the Department of Agricultural Biochemistry arid a Research Assistant in the Department of Medicine. It* June, 1957, i was appointed a United States public Health Service Follow of the National Cancer Institute, j ;neld this position one and a half years while completing the requirements for the degree Doctor of Philosophy,